# US 5,334,537 A · Direct immunoassay using pH-sensitive fluorochromes

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- Number: US 5,334,537 A
- Document ID: US5334537A
- Status: Granted
- Kind: A
- Application number: 07/791,309
- Filed: 1991-11-14
- Granted: 1994-08-02
- Earliest claimed: 1991-11-14
- Assignees: University of Maryland
- Inventors: Cheng S. Lee; Ping Y. Huang
- Web page: https://patentgrep.com/patent/US5334537A

## CPC

- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- Y10S 436/80: Fluorescent dyes, e.g. rhodamine
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The complexing of an antibody-antigen binding pair is determined by observing the change in fluorescence of a pH-sensitive fluorochrome attached to one of the members of the binding pair. When the binding is conducted in a solution having a pH other than the isoelectric point of the antibody, there will be a change in the pH of the microenviromnent surrounding the fluorochrome. This change will correspond to a change in the observed fluorescent intensity. Either member of the binding pair can be labeled, and combined with that member whose presence is suspect, in an immunoassay.

## Claims

### Claim 1

A method of directly detecting complexing of an antigen/antibody binding pair, comprising:
a) preparing a solution of one of said members of said binding pair complexed with a fluorochrome selected from the group consisting of fluorescein, umbelliferones, pyrenes, resorfins, hydroxyesters of aromatic acids, styryl dyes and tetramethyl rhodamine dyes said fluorochrome being complexed with said one of said members of said binding pair at a point spatially proximal to a binding site for said antigen/antibody binding pair in a buffer having a pH other than the isoelectric point of said antibody, and determining the intensity of fluorescence thereof,
b) adding said remaining member of said binding pair to said solution and monitoring the intensity of the fluorescence of said solution,
c) wherein a change in fluorescent intensity in said solution after step b corresponds to the complexing of said antigen with said antibody said solution being free of molecular moieties which affect the intensity of the fluorescence of said fluorochrome, said change in fluorescent intensity being induced by a change in the microenvironment of said fluorochrome induced by said complexing event.

### Claim 2 (depends on 1)

The method of claim 1, wherein said member of said binding pair complexed with said fluorochrome is said antigen.

### Claim 3 (depends on 1)

The method of claim 1, wherein said binding pair member complexed with said fluorochrome is said antibody.

### Claim 4 (depends on 1)

The method of claim 1, wherein said fluorochrome is fluorescein.

### Claim 5 (depends on 1)

The method of claim 1, wherein said binding pair member not complexed with said fluorochrome is contained in an analyte.

### Claim 6 (depends on 1)

The method of claim 1, wherein said fluorochrome is a pH-sensitive fluorochrome.

### Claim 7 (depends on 1)

The method of claim 1, wherein said fluorochrome is an electrostatic potential-sensitive fluorochrome.